Flow cytometry is a powerful tool for the identification of cell populations based on the expression level of target molecules on cells. In keeping with the increasing importance of flow cytometry in biology and medicine, the number and data acquisition power of flow cytometry instruments has expanded greatly in the last few years (Bendall S C et al. A deep profiler's guide to cytometry. Trends in Immunology. 2012. Vol. 33(7):323-32. doi: 10.1016/j.it.2012.02.010; Perfetto S P et al. Seventeen-colour flow cytometry: Unravelling the immune system. Nature Reviews. Vol. 4: 648-655. doi:10.1038/nri1416). Modern flow cytometry is particularly useful for disease diagnostic purposes because it enables simultaneous measurement of up to 20 markers on the inside and surface of each of a very large number of cells in a sample. In particular, differences in antigen expression on small subsets of cells may be informative relative to clinical outcomes such as drug response, disease susceptibility and prognosis. Thus, subsets of cells identified by flow cytometry are frequently compared to find such differences. Specifically, comparisons between a disease sample and control, different genetically modified organisms, or samples that have undergone stimulations provide fundamental information (Gernez Y et al. Blood basophils from cystic fibrosis patients with allergic bronchopulmonary aspergillosis are primed and hyper-responsive to stimulation by aspergillus allergens. Journal of Cystic Fibrosis. 2012. Vol. 11(6):502-510. doi: 10.1016/j.jcf.2012.04.008; Serke S et al. Expression of class I, II, and III epitopes of the CD34 antigen by normal and leukemic hemopoietic cells. Cytometry. 1996. Vol. 26: 154-160; Liu Z et al. Elevated relative fluorescence intensity of CD38 antigen expression on CD8+ T cells is a marker of poor prognosis in HIV infection: results of 6 years of follow-up. Cytometry. 1996. Vol. 26:1-7). Therefore, it is important to have appropriate methods to characterize these differences in a quantitative and useful way. However, while flow instrumentation has improved markedly to meet these needs, there is still a lack of appropriate methods for clinically useful quantitation of differences between subsets of cells in routine and high-throughput analyses.
Flow cytometry users operate with relative fluorescence intensity (FI) values for the cell subset of interest, which makes it almost impossible to directly compare (without normalization on shared control samples) different flow cytometers and even different experiments on the same machine. Flow cytometer settings, in terms of lasers and optical alignment, light collection, optical filters and photodetector sensitivity (Chance J T et al. Instrument-dependent fluorochrome sensitivity in flow cytometric analyses. Cytometry. 1995. Vol. 22(3):232-42) have not been successfully standardized. In addition, different dye conjugates are often available for a given antibody, antibody preparations with the same fluorochrome vary from vendor to vendor, and differences in sample processing (e.g., the incubation time) generate additional variability.
In order to overcome these difficulties, there have been various efforts to quantitate the FI of beads (or cells), that is, to estimate the number of expressed molecules. Traditional methods for estimating the number of expressed molecules on cells, based on the detection of target antigens bound with fluorescently labeled antibodies, assume that the antigen-antibody reaction reaches equilibrium, and hence, that the amount bound correctly reports the amount of antigen on the cell. However, at a minimum, a calibration procedure with carefully prepared reagents is needed to convert the intensity of the fluorescence signal to the number of target antigens (Serke S et al. Quantitative fluorescence flow cytometry: A comparison of the three techniques for direct and indirect immunofluorescence. Cytometry. 1998. Vol. 33(2):179-87). For instance, among the currently marketed technologies, there are three technologies that are well known: Quantum Simply Cellular beads (QSC) designed to bind any fluorochrome-labeled murine monoclonal antibody; Quantitative Immunofluorescence Intensity beads (QIFI kit) for indirect immunofluorescence; and the Quanti-BRITE assay (Schwartz A et al. Development of clinical standards for flow cytometry. Ann N Y Acad Sci. 1993. Vol. 677:28-39. doi:10.1111/j.1749-6632.1993.tb38760.x; Poncelet P et al. Cytofluorometric quantification of cell-surface antigens by indirect immunofluorescence using monoclonal antibodies. Journal of Immunological Methods. 1985. Vol. 85(1):65-74. doi:10.1016/0022-1759(85)90274-1; Davis K A et al. Determination of the number of fluorescent molecules on calibration beads for flow cytometry. U.S. Pat. No. 5,620,842 A. 1997). Although the calibration bead-based technologies seem to be a straightforward and easy-to-use approach for quantitative fluorescence flow cytometry, comparison of these three technologies has revealed their limitations (Serke S et al. Quantitative fluorescence flow cytometry: A comparison of the three techniques for direct and indirect immunofluorescence. Cytometry. 1998. Vol. 33(2):179-87).
The QSC bead-based data were found to be comparable only if they were obtained using a single strictly uniform approach (Denny T N et al. Quantitative determination of surface antibody capacities of immune subset present in peripheral blood of healthy adult donors. Cytometry. 1996. Vol. 26:265-274; Lenkei R et al. Determination of the antibody binding capacity of lymphocyte membrane antigens by flow cytometry in 58 blood donors. Journal of Immunological Methods. 1995. Vol. 183:267-277. doi: 10.1016/0022-1759(95)00064-H). Additionally, the use of the QSC assay with FITC and PE reagents revealed substantially different estimates of cellular binding sites (Serke S et al. Quantitative fluorescence flow cytometry: A comparison of the three techniques for direct and indirect immunofluorescence. Cytometry. 1998. Vol. 33(2):179-87). The use of QIFI calibration kit is restricted since it is marketed with a single manufacturer-defined fluorescent antibody. The Quanti-BRITE assay is specified for use of specially-prepared equimolar (1 antibody molecule:1 PE molecule) reagents only. In general, these approaches are not applicable to labeling with lower affinity antibodies and/or to labeling under non-equilibrium conditions. The choice of calibrator, fluorochrome conjugates and details of sample handling can affect the determination of antigen concentration on beads or cells.
If target sites are very mobile (the surface diffusion of the sites on the cell membrane is fast in comparison with the 3-dimension diffusion of the ligand molecules in the medium) or sufficiently close to each other (the distance between sites are equal or less than the radius of the sites) for some IgG antibodies to bind divalently, the number of effective antibody binding sites will be lower than the number of target antigens. This is a common limitation of the antibody-based methods mentioned above. Thus, special approaches like use on univalent antibodies are needed to resolve this issue.
It has been shown that flow cytometry data for antigen-antibody interactions can be analyzed as a temporal evolution of the cellular fluorescence profile to obtain information on the cellular distribution of the surface antigens, as well as the association and dissociation rate constants per antigen (Orlova D et al. Distribution function approach to study the kinetics of IgM antibodies binding to FcγRIIIb (CD16b) receptors on neutrophils by Flow Cytometry. Journal of Theoretical Biology. 2011. Vol. 290:1-6. doi:10.1016/j.jtbi.2011.08.026; Surovtsev I V et al. Mathematical modeling the kinetics of cell distribution in the process of ligand-receptor binding. Journal Theoretical Biology. 2000. Vol. 206(3):407-17. doi:10.1006/jtbi.2000.2136). However, this information was obtained with the use of calibrators.
The described invention provides a further developed and optimized kinetic approach to antigen quantification on beads and cells which can be applied to both low and high affinity antibodies, under both saturating and non-saturating binding conditions, independent of the conjugated fluorochrome. Instead of using a static calibration system, the mean fluorescence dynamics of a population of interest measured by flow cytometry only are analyzed, in order to evaluate the amount of surface antigens. Experimental data obtained with an LSRII cytometer was fitted by the diffusion-reaction mathematical model for stable binding conditions (the solution for the general case, applied to both low and high affinity antibodies, was described in Orlova D et al. Distribution function approach to study the kinetics of IgM antibodies binding to FcγRIIIb (CD16b) receptors on neutrophils by Flow Cytometry. Journal of Theoretical Biology. 2011. Vol. 290:1-6. doi:10.1016/j.jtbi.2011.08.026) using the Levenberg-Marquardt nonlinear least squares curve-fitting algorithm in order to obtain the number of target antigens per bead/cell. As a result, the binding rate constant for each particular antibody-antigen reaction can be used instead of calibrators in order to quantify antigen molecules per cell using flow cytometry.